EP2945937B1 - Helquat derivatives, preparation thereof, and use thereof as medicaments - Google Patents
Helquat derivatives, preparation thereof, and use thereof as medicaments Download PDFInfo
- Publication number
- EP2945937B1 EP2945937B1 EP14702744.5A EP14702744A EP2945937B1 EP 2945937 B1 EP2945937 B1 EP 2945937B1 EP 14702744 A EP14702744 A EP 14702744A EP 2945937 B1 EP2945937 B1 EP 2945937B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- ddd
- phenanthroline
- rac
- pyrido
- tetrahydroisoquinolino
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Not-in-force
Links
- FMFFTMXAKRYJHA-UHFFFAOYSA-N CC(Cc(ccc1c2)cc1ccc2OC)c1cc[n+](CCc2ccc(CC[n+]3ccccc3-3)c-3c2-2)c-2c1 Chemical compound CC(Cc(ccc1c2)cc1ccc2OC)c1cc[n+](CCc2ccc(CC[n+]3ccccc3-3)c-3c2-2)c-2c1 FMFFTMXAKRYJHA-UHFFFAOYSA-N 0.000 description 1
- NTRIXFQQPKBICR-VOTSOKGWSA-N COc1ccc(/C=C/c2cc[n+](CCc3ccc(CC[n+]4ccc(cccc5)c5c4-4)c-4c3-3)c-3c2)cc1 Chemical compound COc1ccc(/C=C/c2cc[n+](CCc3ccc(CC[n+]4ccc(cccc5)c5c4-4)c-4c3-3)c-3c2)cc1 NTRIXFQQPKBICR-VOTSOKGWSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
Definitions
- the invention relates to new helquat derivatives, preparation thereof, and use thereof as medicaments for treatment of diseases related to increased cellular proliferation.
- the proteins take part mainly in the regulation of cell division and differentiation, in the control of DNA replication fidelity, in the regulation of apoptosis of the damaged cells, in intercellular communication and intracellular signaling pathways [ Hanahan D., Weinberg R. A. (2000), Cell 100, 57-70 ].
- Malignant cells unlike benign cells, have the ability to penetrate into the surrounding healthy tissue (invasiveness). Cancer cells are can be released from the original tumor and spread through the bloodstream or lymphatic system to distant parts of the body to form new tumors (metastatic process) [ Nguyen D. X. et al. (2009), Nat. Rev. Cancer 9, 274-284 ].
- the aim of anticancer therapy is to selectively induce apoptosis in the undesirable cancer cells, while not affecting the surrounding healthy tissue.
- Cytotoxic therapeutics act through DNA damage or microtubule damage and their specificity towards tumor cells in human body is due to their ability to selectively kill fast-proliferating cells. This selectivity can be determined by their cytostatic effects in cell culture in vitro [ Chabner B. A., Roberts T. G. (2005), Nat. Rev. Cancer 5, 65-72 ; Lüllmann H. et al. (2005), Farmakologie a toxikologie, Grada, 15th editi on].
- tumor cells are derived from cells of a host organism is a limiting factor for achieving the maximal selectivity of the cytotoxic effect.
- the sensitivity of cancer cells towards treatment is determined by the growth fraction of a tumor (the ratio of proliferating and non-proliferating tumor cells), the site of action of the cytostatic agent within the cell cycle, and the natural and the acquired resistence of the tumor cells against the cytostatics.
- WO03/097642 discloses polycyclic carboxamides for use in the treatment of cellular proliferative disorders such as cancer.
- the present invention opens a straightforward way for obtaining novel compounds, structurally belonging to the helquat family and useful as therapeutics for diseases related to increased cellular proliferation.
- a disadvantage of the heretofore described synthesis of these compounds was a limited variability due to the need for assembling each compound in a multistep synthesis de novo.
- the present invention overcomes this disadvantage, as it introduces not only novel helquat derivatives, but also method of preparation thereof by one-step diversification of methyl-substituted helquats using Knoevenagel condensation with substituted or non-substituted arylaldehydes.
- Pharmaceutically acceptable salts include salts with alkali metals, salts with inorganic or organic anions, and further salts suitable for physiological application.
- the pharmaceutically acceptable salts of compounds of general formula I may be salts with anions derived from inorganic or organic acids.
- a person skilled in the art would be able to determine which salts are pharmaceutically acceptable, in particular salts having one or more physical properties such as enhanced pharmaceutical stability in differing temperatures and humidities, a desirable solubility in water or oil, or non-toxicity.
- Suitable pharmaceutically acceptable salts of the compounds of the present invention in particular include anions derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids.
- inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids
- organic acids such as acetic
- Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.
- suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, pamoate, methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate,
- the object of the invention are also helquat derivatives of the following formulae:
- object of the invention are helquat derivatives of general formula I according to this invention for use as medicaments.
- Another object of the invention are helquat derivatives of general formula I according to this invention for use as medicaments in the treatment of diseases related to increased cellular proliferation.
- a further object of the invention are helquat derivatives of general formula I according to this invention for use in the treatment of oncologic diseases.
- a solvent selected from a group comprising methanol, ethanol, acetonitrile, dimethylsulfoxide and dimethylformamide, is used as the organic solvent.
- a further object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt.
- Yet another object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt as an active ingredient and optionally at least one pharmaceutically acceptable carrier, filler, or diluent.
- a further object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt as an active ingredient, for use in the treatment of diseases related to increased cellular proliferation.
- Another object of the present invention is use of helquat derivatives of general formula I according to this invention or their pharmaceutically acceptable salts for the preparation of a medicament for the treatment of diseases related to increased cellular proliferation.
- Yet another object of the present invention is use of helquat derivatives of general formula I according to this invention or their pharmaceutically acceptable salts for the preparation of a medicament for the treatment of oncologic diseases.
- helquats we regarded the helquats to be selectively toxic against cancer cell lines if they effected 50% decrease of metabolic activity (proliferation) of the treated cells in concentration up to 50 ⁇ mol.l -1 (i.e. IC 50 ⁇ 50 ⁇ mol.l -1 ). Helquats showing IC 50 higher than 150 ⁇ mol.l -1 for a given cell line were classified as non-toxic for that cell line. Efficient growth inhibition of the cancer cell lines was assessed in micromolar concentrations, even though only a minimal effect on normal cells was detected. Therefore, helquats are useful as selective therapeutics for treatment of hyperproliferation of mammalian cells or as antimitotic and apototic drugs, mainly in anticancer therapy.
- Notation used in the NMR spectra s (singlet), d (dublet), t (triplet), q (quartet), m (multiplet), b (broad signal, e.g., bdt denotes broad dublet of triplets).
- IR infrared spectra
- Racemic helquat B (70 mg, 0.103 mmol), 9-julolidinecarbaldehyde (147 mg, 0.725 mmol), pyrrolidine (0.100 ml), and dry methanol (7 ml) were placed into a flask and the mixture was stirred under argon for 2.5 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 41 mg (0.048 mmol, 47% yield) of Knoevenagel condensation product 12 as a violet crystalline solid.
- Cell lines derived from various cancers are key for complex validation of antiproliferative effects of test compounds in vitro. Testing for evaluation of effects of the studied helquat derivatives was performed with 6 model cancer cell lines and 2 normal (healthy) cell lines of diverse histological origin. All the cell lines listed below were cultivated under conditions for their optimal growth in the given medium in plastic bottles or plastic Petri dishes of various sizes (TPP, BD Biosciences) at 37 °C, 5% CO 2 and 95% air humidity. HUVEC cells were obtained from BD Biosciences. All other cell lines were obtained from ATCC/LGC Standards (American Type Cell Collection).
- CCRF-CEM (cat. no. ATCC CCL-119)
- Suspension cell line CCRF-CEM is a permanent in vitro culture of acute lymphoblastic leukemia.
- CCRF-CEM cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, cat. no. R8758) with addition of 2 mmol.l -1 glutamine (Invitrogen, cat. no. 35050-038), 10% fetal bovine serum (FBS, Sigma-Aldrich, cat. no. F9665), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, cat. no. P0781). Passaging was performed 2-3x a week. Population doubling time of CCRF-CEM cell line was 20 h under the cultivation conditions used.
- Suspension cell line HL-60 is a permanent in vitro culture of acute promyelocytic leukemia.
- HL-60 cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, see above) with addition of 2 mmol.l -1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed 2-3x a week. Population doubling time of HL-60 cell line was 23 h under the cultivation conditions used.
- MOLT-4 (cat. no. ATCC CRL-1582)
- MOLT-4 is a permanent in vitro culture of acute lymfoblastic leukemia.
- MILT-4 cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, see above) with addition of 2 mmol.l -1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed 2-3x a week. Population doubling time of MOLT-4 cell line was 25 h under the cultivation conditions used.
- Adherent cell line HeLa is a permanent in vitro culture derived from cervical cancer.
- HeLa cell line was cultivated in RPMI 1640 medium (Dutch modification) (Sigma-Aldrich, cat. no. R7638) with addition of 2 mmol.l -1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week. After washing of cells with sterile PBS the cells were released by 0.25% trypsin/EDTA (Sigma-Aldrich, cat. no.
- Hep G2 (cat. no. ATCC HB-8065)
- Adherent cell line Hep G2 is a permanent in vitro culture derived from liver tissue of a patient with hepatocellular carcinoma.
- Hep G2 cell line was cultivated in DMEM medium (Sigma-Aldrich, cat. no. M4528) with addition of 2 mmol.l -1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week.
- Adherent cell line LoVo is a permanent in vitro culture derived from tissue of a patient with colorectal carcinoma.
- LoVo cell line was cultivated in DMEM medium (Sigma-Aldrich, cat. no. D8437) with addition of 2 mmol.l -1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 ⁇ g/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week.
- HUVEC (BD Biosciences, cat. no. 354151)
- cytotoxity test for cell viability assessment based on ATP quantification in cell lysates was used (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, cat. no. G7571).
- ATP in cell lysates is detected using luciferase reaction.
- Luminescence intensity correlates with the ATP level and thus with quantity of metabolically active (viable) cells.
- IC 50 value serves as the output, that is the concentration of the test compound which leads to 50 % reduction in number of viable cells as compared to control, untreated population. To this end, IC 50 value reflects efficiency of the test compound with regard to the given cell line.
- volume of solvent equal to the volume of added compound solutions was added. After 72 h of incubation with the test compounds viability was determined according to the manufacturer's instructions. In short: microtiter well plate was left 30 min to reach the room temperature after it has been removed from the incubator. Then, into each well, 100 ⁇ l of the prepared detection agent was added. Next, the well plate was shaken for 2 min to complete the full lysis. Next, after the well had been left in the dark for 15 min, the luminescence was measured in each well using luminometer (Tecan Genios, Austria).
- IC 50 value represents concentration of the test compound, which leads to 50% reduction of viable cells (inhibition of cell growth) after 72 h of the treatment.
- Each helquat concentration was tested in triplicates in a IC 50 quantification and at least 3 independent IC 50 quantification experiments were performed.
- IC 50 values over 100 ⁇ mol.l -1 were obtained by extrapolation of data measured in concentration range 0 - 100 ⁇ mol.l -1 for the given helquat.
- the invention is useful in pharmaceutical industry and medicine for the treatment of diseases related to increased cellular proliferation, for example tumor growth.
Description
- The invention relates to new helquat derivatives, preparation thereof, and use thereof as medicaments for treatment of diseases related to increased cellular proliferation.
- Original compounds useful in cancer therapy are subject of interest in industrial and academic laboratories [Avendano, C., Menendez, J. C. (2008) Medicinal Chemistry of Anticancer Drugs, Elsevier Science, 1st edition].
Malignant tumor diseases are the most frequent cause of death [Siegel R. et al. (2012), CA Cancer J. Clin. 62, 10-29]. The uncontrolled cellular growth is linked to inherited genetic factors as well as environmental factors. For initiation and development of a malignant disease, the accumulation of several various genetic or epigenetic changes is necessary. This leads to transformation of a healthy cell into a fully malignant phenotype [Stratton M. R. (2011), Science 331, 1553-1558]. Cumulation of gene mutations leads to perturbations in normal functioning of proteins encoded by these genes. The proteins take part mainly in the regulation of cell division and differentiation, in the control of DNA replication fidelity, in the regulation of apoptosis of the damaged cells, in intercellular communication and intracellular signaling pathways [Hanahan D., Weinberg R. A. (2000), Cell 100, 57-70]. Malignant cells, unlike benign cells, have the ability to penetrate into the surrounding healthy tissue (invasiveness). Cancer cells are can be released from the original tumor and spread through the bloodstream or lymphatic system to distant parts of the body to form new tumors (metastatic process) [Nguyen D. X. et al. (2009), Nat. Rev. Cancer 9, 274-284]. - The aim of anticancer therapy is to selectively induce apoptosis in the undesirable cancer cells, while not affecting the surrounding healthy tissue. Cytotoxic therapeutics act through DNA damage or microtubule damage and their specificity towards tumor cells in human body is due to their ability to selectively kill fast-proliferating cells. This selectivity can be determined by their cytostatic effects in cell culture in vitro [Chabner B. A., Roberts T. G. (2005), Nat. Rev. Cancer 5, 65-72; Lüllmann H. et al. (2005), Farmakologie a toxikologie, Grada, 15th edition].
- The fact that tumor cells are derived from cells of a host organism is a limiting factor for achieving the maximal selectivity of the cytotoxic effect. The sensitivity of cancer cells towards treatment is determined by the growth fraction of a tumor (the ratio of proliferating and non-proliferating tumor cells), the site of action of the cytostatic agent within the cell cycle, and the natural and the acquired resistence of the tumor cells against the cytostatics.
WO03/097642 - The present invention opens a straightforward way for obtaining novel compounds, structurally belonging to the helquat family and useful as therapeutics for diseases related to increased cellular proliferation.
- Recently, papers describing synthesis of helical extended diquats (helquats) have been published [Adriaenssens et al. (2009), Chem. Eur. J. 15, 1072-1076; Severa et al. (2010), Tetrahedron 66, 3537-3552; Vávra et al. (2012), Eur. J. Org. Chem. 489-499]. They represent a new and therefore unexplored class of compounds with dicationic helical skeleton. The basic helquat skeletons described heretofore are composed so that each skeleton contains two quaternary N-heteroaromatic units which introduce two positively charged centers into the system, e.g. in the form of pyridinium, quinolinium, or isoquinolinium cationic moieties. Hence, a typical helquat arrangement is associated with dicationicity and with helical chirality at the same time. This combination has not been studied before in the context of small aromatic organic molecules.
- A disadvantage of the heretofore described synthesis of these compounds was a limited variability due to the need for assembling each compound in a multistep synthesis de novo. The present invention overcomes this disadvantage, as it introduces not only novel helquat derivatives, but also method of preparation thereof by one-step diversification of methyl-substituted helquats using Knoevenagel condensation with substituted or non-substituted arylaldehydes.
- The object of the present invention are helquat derivatives of the general formula I
- substituents R1 and R2 are independently selected from the group comprising H and C1 to C3 alkyl,
- up to three of S1,2, S1',2', S3,4 and S3',4' are present,
- each of S1,2, S1',2', S3,4 and S3',4' independently represents a linker consisting of a bivalent hydrocarbon chain having 3-6 carbon atoms, preferably hydrocarbon chain having 4 carbon atoms, more preferably hydrocarbon chain having 4 carbon atoms and two double bonds, and
- one or two atoms selected from the carbon atoms with the descriptor 2, 4, 2', and 4' (the carbon atom must be free of the S-linker as would be apparent to a person skilled in the art) are substituted with a substituent R3 of general formula II
- T1 and T2 are linkers that bridge atoms N5 with C8 and N5' with C8', wherein T1 and T2 independently represent a bivalent hydrocarbon chain having 2-5 carbon atoms, preferably 2 or 3 carbon atoms,
wherein
aryl is a hydrocarbon group containing 6 to 16 carbon atoms, preferably 6 to 12 carbon atoms, and at least one aromatic ring, wherein the aryl can be unsubstituted or substituted with 1 to 5 substituents, selected from a group comprising C1 to C6 alkyl, C1 to C6 halogenoalkyl, C1 to C12 alkoxy, C6 to C16 aryloxy, benzyloxy, C1 to C6 alkylthio, arylthio, halogeno, -OH, -SH, -NH2, C1 to C6 alkylamino, arylamino, C1 to C6 acylamino, -CN, nitro, and -COORn, wherein Rn is hydrogen or C1 to C6 alkyl or C6 to C16 aryl;
and anions (X1)- and (X2)- independently represent anions of pharmaceutically acceptable salts of general formula I. - Pharmaceutically acceptable salts include salts with alkali metals, salts with inorganic or organic anions, and further salts suitable for physiological application.
- The pharmaceutically acceptable salts of compounds of general formula I may be salts with anions derived from inorganic or organic acids. A person skilled in the art would be able to determine which salts are pharmaceutically acceptable, in particular salts having one or more physical properties such as enhanced pharmaceutical stability in differing temperatures and humidities, a desirable solubility in water or oil, or non-toxicity.
- Suitable pharmaceutically acceptable salts of the compounds of the present invention in particular include anions derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.
Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, pamoate, methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, 13-hydroxybutyrate, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, and undecanoate. - The object of the invention are also helquat derivatives of the following formulae:
- (rac)-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (rac)-1,
- (M)-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (M)-1,
- (P)-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (P)-1,
- (rac)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (rac)-7,
- (M)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (M)-7,
- (P)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (P)-7,
- (rac)-(E)-11-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (rac)-12,
- (rac)-(E)-13-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-4,5,8,9-tetrahydro-isoquinolino[2,1-k]pyrido[2,1-a][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate, i.e. (rac)-20,
- (rac)-(E)-19-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-8,9,10,13,14,15-hexahydropyrido[1"',2"':1",2"]azepino[4",3":5',6']benzo[1',2':3,4]-azepino[2,1-a]isoquinoline-7,16-diium trifluoromethanesulfonate, i.e. (rac)-25,
- 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-6,7,10,11-tetrahydrodipyrido[2,1-a:1',2'-k][2,9]phenanthroline-5,12-diium trifluoromethanesulfonate, i.e. 30,
- (M)-(E)-13-(4-methoxystyryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino[1,2-a]pyrido-[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethansulfonate, i.e. (M)-6,
- (rac)-4,15-bis((E)-4-(dimethylamino)styryl)-6,7,8,11,12,13-hexahydrodipyrido-[1,2-a:1',2'-a']benzo[2,1-c:3,4-c']bisazepinediium trifluoromethanesulfonate, i.e. (rac)-41.
- Furthermore, object of the invention are helquat derivatives of general formula I according to this invention for use as medicaments.
- Another object of the invention are helquat derivatives of general formula I according to this invention for use as medicaments in the treatment of diseases related to increased cellular proliferation.
- A further object of the invention are helquat derivatives of general formula I according to this invention for use in the treatment of oncologic diseases.
- Another object of the invention is a method of preparation of helquat derivatives of general formula I according to this invention, in which a starting helquat bearing reactive methyl group, corresponding to helquat of general formula I wherein R3 is methyl and other substituents are as described above, is reacted with substituted or unsubstituted arylaldehyde which can be R4-CHO or R4-CH=CH-CHO in the presence of a base, preferably pyrrolidine or piperidine, and in an organic solvent, and the resulting product is isolated by precipitation from the solution.
- In a preferred embodiment of the method of preparation, a solvent, selected from a group comprising methanol, ethanol, acetonitrile, dimethylsulfoxide and dimethylformamide, is used as the organic solvent.
- A further object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt.
- Yet another object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt as an active ingredient and optionally at least one pharmaceutically acceptable carrier, filler, or diluent.
- A further object of the invention is a pharmaceutical agent, containing at least one helquat derivative of general formula I according to this invention or its pharmaceutically acceptable salt as an active ingredient, for use in the treatment of diseases related to increased cellular proliferation.
- Another object of the present invention is use of helquat derivatives of general formula I according to this invention or their pharmaceutically acceptable salts for the preparation of a medicament for the treatment of diseases related to increased cellular proliferation.
- Yet another object of the present invention is use of helquat derivatives of general formula I according to this invention or their pharmaceutically acceptable salts for the preparation of a medicament for the treatment of oncologic diseases.
- Currently, there are various clinically used cytostatics. However, therapeutic results in the treatment of patients with malignant disease still remain unsatisfactory and require further search for novel more efficient compounds with lower toxicity against normal, healthy cells, which have a lower ability to induce resistance of the target cells. Therefore, apart from the search for new treatment modalities, the development of compounds targeted at cells with increased proliferation continues to be very important. Compounds with a wide spectrum of activities and a high selectivity index, e.g., against cancer cells, and exhibiting fewer undesired side effects after application are sought. From a commercial point of view, easy preparation/synthesis of such compounds (e.g., helquat derivatives) is important. Their isolation can be performed without the need for using chromatography, which is particularly advantageous from the industrial point of view.
- In tests with six model cancer cell lines, the newly prepared helquat derivatives showed considerable reduction of viability (proliferation) of the cells, while cytotoxicity of these compounds against two non-cancer cell lines was significantly lower or was not detectable at all, see Table 1. Sensitivity of the individual cell lines depended on the cell type; a higher effect on faster proliferating leukemic cell line as opposed to cell lines derived from selected types of solid tumors, was proved.
- In this invention, we regarded the helquats to be selectively toxic against cancer cell lines if they effected 50% decrease of metabolic activity (proliferation) of the treated cells in concentration up to 50 µmol.l-1 (i.e. IC50 < 50 µmol.l-1). Helquats showing IC50 higher than 150 µmol.l-1 for a given cell line were classified as non-toxic for that cell line. Efficient growth inhibition of the cancer cell lines was assessed in micromolar concentrations, even though only a minimal effect on normal cells was detected. Therefore, helquats are useful as selective therapeutics for treatment of hyperproliferation of mammalian cells or as antimitotic and apototic drugs, mainly in anticancer therapy.
- The invention is hereinafter illustrated by the following examples, which should not be construed as further limiting.
- The numerical values of chemical shifts in NMR spectra are given in ppm.
- Notation used in the NMR spectra: s (singlet), d (dublet), t (triplet), q (quartet), m (multiplet), b (broad signal, e.g., bdt denotes broad dublet of triplets).
- Abbreviations in description of infrared spectra (IR): sh (shoulder in absorption band). The following abbreviations describe intensity of IR spectra bands vs (very strong), s (strong), m (medium), w (weak), vw (very weak).
- Where the signal assignments in the NMR spectra are indicated, the numbering scheme used in the corresponding structural formula is arbitrary.
-
- ESI
- electrospray
- m.p.
- melting point
- TfO-
- trifluoromethanesulfonate anion
- PBS
- phosphate buffered saline
- EDTA
- ethylenediaminetetraacetic acid
- ATP
- adenosine triphosphate
- Structures A to I of starting helquats are as follows:
- Starting racemic helquat A (144 mg, 0.213 mmol), 4-dimethylaminobenzaldehyde (41 mg, 0.275 mmol), pyrrolidine (0.20 ml, 2.44 mmol), and dry methanol (2.5 ml) were placed into a 10 ml flask and the resulting mixture was stirred under argon for 5 h at room temperature. Progress of the reaction was monitored by thin layer chromatography. Crude product was precipitated from the reaction mixture by addition of diethylether (90 ml). The suspension was centrifuged and the liquid was separated from the solid pellet. The solids were dissolved in methanol (5 ml) and the pure product was precipitated by addition of diethylether (40 ml). Then, centrifugation of this suspension, removal of the liquid, and drying of the solid product under vacuum of an oil pump led to 121 mg (0.150 mmol, 70% yield) of Knoevenagel condensation (rac)-1 as a dark red crystalline compound.
- Starting racemic helquat A (58 mg, 0.085 mmol), 6-methoxy-2-naphtaldehyde (20 mg, 0.110 mmol), pyrrolidine (0.080 ml, 0.958 mmol), and dry methanol (1 ml) were placed into a flask and the mixture was stirred under argon for 20 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 45 mg (0.053 mmol, 62% yield) of Knoevenagel condensation product (rac)-7 as an orange crystalline solid.
13C NMR (151 MHz, acetonitrile-d3): 17.13 (C-40), 17.21 (C-41), 26.12 (C-15), 26.65 (C-8), 54.61 (C-16), 56.18 (C-7), 56.29 (C-39), 107.27 (C-32), 120.60 (C-34), 121.44 (C-3), 121.94 (C-27), 123.33 (C-10), 125.03 (C-30), 125.49 (C-19), 126.57 (C-5), 126.88 (C-26), 127.82 (C-11), 128.64 (C-31), 128.73 (C-23), 129.01 (C-20), 129.53 (C-38), 131.09 (C-29), 131.09 (C-36), 131.32 (C-35), 132.80 (C-22), 136.50 (C-21), 137.02 (C-37), 137.51 (C-18), 138.64 (C-13), 139.78 (C-25), 141.44 (C-14), 142.25 (C-28), 142.33 (C-12), 142.70 (C-9), 145.54 (C-2), 147.32 (C-6), 151.75 (C-24), 153.89 (C-3), 160.34 (C-33).
IČ (KBr): v (cm-1) 518 m, 573 w, 638 s, 679 vw, 818 w, 755 w, 980 w, 1030 s, 1117 w, 1164 s, 1224 m, 1265 vs, 1352 w, 1381 w, 1393 w, 1411 vw, 1439 w, 1484 m, 1508 w, 1552 w, 1573 w, 1602 s, 1629 m, 2843 vw.
HRMS (ESI) m/z: [(M-CF3SO3 -)+] (C40H34F3N2O4S) calculated: 695.2186, found: 695.2181; [(M-2CF3SO3 -)2+] (C39H34N2O) calculated: 273.1330, found: 273.1330. - Starting helquat E (23 mg, 0.038 mmol), (E)-3-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)acrylaldehyde (17 mg, 0.076 mmol), pyrrolidine (0.038 ml, 0.454 mmol), and dry methanol (4 ml) were placed into a flask and the mixture was stirred under argon for 1 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 23 mg (0.029 mmol, 76% yield) of Knoevenagel condensation product 31 as a dark blue solid.
- Racemic helquat B (70 mg, 0.103 mmol), 9-julolidinecarbaldehyde (147 mg, 0.725 mmol), pyrrolidine (0.100 ml), and dry methanol (7 ml) were placed into a flask and the mixture was stirred under argon for 2.5 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 41 mg (0.048 mmol, 47% yield) of Knoevenagel condensation product 12 as a violet crystalline solid.
IČ (KBr): v (cm-1) 517 m, 573 w, 638 s, 1030 vs, 1163 w, 1484 m, 1524 w, 1553 s, 1588 w, 1627 b, 3074 w.
HRMS (ESI) m/z: [(M-CF3SO3 -)+] (C41H39F3N3O3S) calculated: 710.2659; found: 710.2653.
Elem. analysis: C42H39F6N3O6S2, calculated: C (58.66), H (4.57), F (13.26), N (4.89), S (7.46), found: C (58.42), H (5.08), F (12.92), N (4.40), S (7.18). - Racemic helquat G (60 mg, 0.094 mmol), 4-(dimethylamino)benzaldehyde (174 mg, 1.17 mmol), piperidine (0.111 ml, 1.12 mmol), and dry methanol (2 ml) were placed into a flask and the mixture was stirred under argon for 72 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 79 mg (0.088 mmol, 94% yield) of Knoevenagel condensation product (rac)-41 as a dark red crystalline solid.
- Starting non-racemic helquat (M)-A (35 mg, 0.052 mmol), 4-dimethylaminobenzaldehyde (15 mg, 0.103 mmol), pyrrolidine (0.052 ml, 0.621 mmol), and dry methanol (1 ml) were placed into a flask and the mixture was stirred under argon for 1 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 36 mg (0.044 mmol, 85% yield) of Knoevenagel condensation product (M)-1 as a dark red crystalline solid.
m.p. = 264-266°C. 1H NMR (600 MHz, acetone-d6): 2.52 (s, 3H), 2.55 (s, 3H), 3.08 (s, 6H), 3.23-3.31 (m, 1H), 3.34-3.42 (m, 1H), 3.64-3.73 (m, 2H), 5.01-5.08 (m, 1H), 5.10-5.19 (m, 2H), 5.30-5.34 (m, 1H), 6.48 (d, J= 16.0 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H), 7.13 (d, J= 16.0 Hz, 1H), 7.15 (d, J= 1.9 Hz, 1H), 7.38 (d, J= 8.9 Hz, 2H), 7.66 (dd, J= 1.9, 6.7 Hz, 1H), 7.70-7.74 (m, 1H), 7.87-7.91 (m, 1H), 8.00-8.03 (m, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.47 (d, J = 6.7 Hz, 1H), 8.69 (d, J= 6.7 Hz, 1H), 9.00 (d, J = 6.7 Hz, 1H). 13C NMR (151 MHz, acetone-d6): 16.9; 17.0; 26.3; 26.7; 40.1; 53.9; 56.2; 112.6; 117.1; 120.0; 123.4; 123.4; 125.1; 126.2; 126.7; 128.2; 128.8; 128.9; 131.4; 132.4; 136.1; 137.8; 138.5; 139.7; 140.7; 142.1; 142.2; 143.0; 144.8; 146.7; 151.8; 153.3; 154.6. IČ (film): v (cm-1) 517 w, 573 w, 637 m, 757 vw, 820 w, 944 w, 975 w, br, 1030 s, 1110 m, 1159 s, 1224 m, 1255 vs, 1335 m, 1369 m, 1411 w, 1509 m, 1529 m, 1547 m, 1571 vs, 1630 w. HRMS (ESI) m/z: [(M-CF3SO3 -)+] (C37H35F3N3O3S) calculated: 658.2346, found: 658.2331. - Starting non-racemic helquat (M)-A (51 mg, 0.075 mmol), 6-methoxy-2-naphthaldehyde (57 mg, 0.301 mmol), pyrrolidine (0.188 ml, 2.256 mmol) and dry methanol (2 ml) were placed into a flask and the mixture was stirred under argon for 1 h at room temperature. The reaction progress was monitored by thin layer chromatography. Crude product was isolated by a method analogous to that described in Example 1. This led to 49 mg (0.058 mmol, 77% yield) of Knoevenagel condensation product ( M )-7 as an orange crystalline solid.
1H NMR (400 MHz, acetonitrile-d3): 2.53 (s, 6H), 3.07-3.25 (m, 2H), 3.53-3.63 (m, 2H), 3.93 (s, 3H), 4.79-4.98 (m, 3H), 5.05-5.12 (m, 1H), 6.79 (d, J = 16.3 Hz, 1H), 6.93 (d, J = 1.8 Hz, 1H), 7.08 (d, J= 16.3 Hz, 1H), 7.21-7.25 (m, 1H), 7.30 (d, J= 2.4 Hz, 1H), 7.56-7.67 (m, 3H), 7.78-7.90 (m, 5H), 8.04 (d, J = 8.2 Hz, 1H), 8.36 (d, J= 6.7 Hz, 1H), 8.50 (d, J = 6.6 Hz, 1H), 8.72 (d, J = 6.7 Hz, 1H). IČ (film): v (cm-1) 518 w, 637 s, 756 w, 819 w, 858 w, 978 w, br, 1029 s, 1158 s, 1224 m, 1256 vs, 1352 w, 1393 w, 1434 w, 1483 m, 1509 w, 1551 w, 1573 w, 1600 s, 1628 w. HRMS (ESI) m/z: [(M-CF3SO3 -)+] (C40H34F3N2O4S) calculated: 695.2186, found: 695.2180. - According to procedure in Example 7, reaction of 35 mg of non-racemic helquatu (P)-A and 15 mg of 4-dimethylaminobenzaldehyde gave analogously 33 mg of non-racemic derivative (P)-1 as a dark red crystalline solid (80% yield).
- According to procedure in Example 8, reaction of non-racemic helquat (P)-A and 6-methoxy-2-naphthaldehyde gave analogously non-racemic derivative (P)-7.
-
-
-
-
-
-
-
-
-
-
-
-
- 1H NMR (400 MHz, acetone-d6): 1.84 -1.97 (m, 4H), 2.37 - 2.94 (m, 10H), 3.03 - 3.21 (m, 2H), 3.28 - 3.35 (m, 4H), 4.54 - 4.67 (m, 1H), 4.78 - 4.95 (m, 2H), 5.18 (dd, J= 13.7, 6.4 Hz, 1H), 6.84 (d, J= 15.9 Hz, 1H), 7.07 (s, 2H), 7.37 (d, J= 2.0 Hz, 1H), 7.60 (d, J= 15.9 Hz, 1H), 7.83 (s, 2H), 7.93 (dd, J = 8.0, 1.2 Hz, 1H), 8.07 (dd, J = 6.9, 2.1 Hz, 1H), 8.18 (ddd, J = 7.7, 6.2, 1.4 Hz, 1H), 8.48 (td, J = 7.9, 1.4 Hz, 1H), 8.82 (d, J= 6.9 Hz, 1H), 9.34 (dd, J = 6.2, 1.3 Hz, 1H).
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
- Influence of the test compounds on viability (proliferation) of cell lines used was investigated in concentration range 0 - 100 µmol.l-1.
- Cell lines derived from various cancers are key for complex validation of antiproliferative effects of test compounds in vitro. Testing for evaluation of effects of the studied helquat derivatives was performed with 6 model cancer cell lines and 2 normal (healthy) cell lines of diverse histological origin. All the cell lines listed below were cultivated under conditions for their optimal growth in the given medium in plastic bottles or plastic Petri dishes of various sizes (TPP, BD Biosciences) at 37 °C, 5% CO2 and 95% air humidity. HUVEC cells were obtained from BD Biosciences. All other cell lines were obtained from ATCC/LGC Standards (American Type Cell Collection).
- Suspension cell line CCRF-CEM is a permanent in vitro culture of acute lymphoblastic leukemia. CCRF-CEM cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, cat. no. R8758) with addition of 2 mmol.l-1 glutamine (Invitrogen, cat. no. 35050-038), 10% fetal bovine serum (FBS, Sigma-Aldrich, cat. no. F9665), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, cat. no. P0781). Passaging was performed 2-3x a week. Population doubling time of CCRF-CEM cell line was 20 h under the cultivation conditions used.
- Suspension cell line HL-60 is a permanent in vitro culture of acute promyelocytic leukemia. HL-60 cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, see above) with addition of 2 mmol.l-1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed 2-3x a week. Population doubling time of HL-60 cell line was 23 h under the cultivation conditions used.
- Suspension cell line MOLT-4 is a permanent in vitro culture of acute lymfoblastic leukemia. Linie MOLT-4 cell line was cultivated in RPMI 1640 medium (Sigma-Aldrich, see above) with addition of 2 mmol.l-1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed 2-3x a week. Population doubling time of MOLT-4 cell line was 25 h under the cultivation conditions used.
- Adherent cell line HeLa is a permanent in vitro culture derived from cervical cancer. HeLa cell line was cultivated in RPMI 1640 medium (Dutch modification) (Sigma-Aldrich, cat. no. R7638) with addition of 2 mmol.l-1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week. After washing of cells with sterile PBS the cells were released by 0.25% trypsin/EDTA (Sigma-Aldrich, cat. no. T4049) at 37 °C for 2 minutes. The trypsin was inactivated by addition of 2 volumes of complete medium, cells were then centrifuged (250 x g, 5 min), the cell pellet was resuspended, and transferred in the amount needed into a new cultivation bottle with fresh medium. Population doubling time of MOLT-4 cell line was 25 h under the cultivation conditions used.
- Adherent cell line Hep G2 is a permanent in vitro culture derived from liver tissue of a patient with hepatocellular carcinoma. Hep G2 cell line was cultivated in DMEM medium (Sigma-Aldrich, cat. no. M4528) with addition of 2 mmol.l-1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week. After washing of cells with sterile PBS, the cells were released by 0.25% trypsin/EDTA (Sigma-Aldrich, see above) at 37 °C for 2 minutes. The trypsin was inactivated by addition of 2 volumes of complete medium, cells were then centrifuged (250 x g, 5 min), resuspended, and transferred in the amount needed into a new cultivation bottle with fresh medium. Population doubling time of Hep G2 cell line was 28 h under the cultivation conditions used.
- Adherent cell line LoVo is a permanent in vitro culture derived from tissue of a patient with colorectal carcinoma. LoVo cell line was cultivated in DMEM medium (Sigma-Aldrich, cat. no. D8437) with addition of 2 mmol.l-1 glutamine (Invitrogen, see above), 10% fetal bovine serum (FBS, Sigma-Aldrich, see above), 100 IU/ml peniciline, 100 µg/ml streptomycine (Sigma-Aldrich, see above). Passaging was performed twice a week. After washing of cells with sterile PBS, the cells were released by 0.25% trypsin/EDTA (Sigma-Aldrich, see above) at 37 °C for 2 minutes. The trypsin was inactivated by addition of 2 volumes of complete medium, cells were then centrifuged (250 x g, 5 min), resuspended, and transferred in the amount needed into a new cultivation bottle with fresh medium. Population doubling time of LoVo cell line was 39 h under the cultivation conditions used.
- Normal human endothelial cells from umbilical vein are adherent cells cultivated in plastic Petri dishes coated with collagen I. Into the cultivation medium (E-STIM™; BD Biosciences, cat. co. 355054) already containing 2% FBS (fetal bovine serum), hydrocortisone and heparin the following two further ingredients were added: epidermal growth factor (EGF, 5 µg) and mixture of factors facilitating growth of endothelial cells (ECGS, 100 mg). The cultivation medium used did not contain antibiotics. The cells were passaged after reaching 90% confluency by dissociation using 0.25% solution of trypsin/EDTA (1 ml; 3 min; 37 °C). After that 5 ml of complete cultivation medium E-STIM™ was added and the cells were centrifuged (180 x g, 7 min), the pellet was resuspended, and transferred in the amount needed into a new Petri dish with fresh medium. The cells were used in experiments between the 3rd and the 8th passage after defreezing. Population doubling time of HUVEC cell line was 38 h under the cultivation conditions used.
- Normal human dermal fibroblasts were cultivated without antibiotics in basal medium for fibroblasts (ATCC/LGC Standards, cat. no. PCS-201-030) with added "Fibroblast Growth Kit-Low serum" (ATCC/LGC Standards, cat. no. PCS-201-041) containing 2% FBS, L-glutamine, hydrocortisone, ascorbic acid, rh FGF beta, rh insulin. When 90% confluency was reached the fibroblasts were washed with sterile PBS (5 ml) and dissociated by addition of 0.25% solution of trypsin/EDTA (2 ml; 2 min; 37 °C). After that 5 ml of cultivation medium for fibroblasts was added and the cells were centrifuged (180 x g, 7 min), the cell pellet was resuspended, and transferred in the amount needed into a new cultivation bottle with fresh medium. The same cell release procedure was applied when the cells were to be used in experiments. The cells were used in experiments between the 3rd and the 15th passage after defreeezing. Population doubling time of NHDF-Ad cell line was 33 h under the cultivation conditions used.
- In order to test the sensitivity of the cell lines towards the studied helquats, cytotoxity test for cell viability assessment based on ATP quantification in cell lysates was used (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, cat. no. G7571). In this test, ATP in cell lysates is detected using luciferase reaction. Luminescence intensity correlates with the ATP level and thus with quantity of metabolically active (viable) cells. IC50 value serves as the output, that is the concentration of the test compound which leads to 50 % reduction in number of viable cells as compared to control, untreated population. To this end, IC50 value reflects efficiency of the test compound with regard to the given cell line.
- Cells in the exponential growth phase were seeded into a 96-well microtiter plate were plated at 3000 cells per well. Each well contained 90 µl of cell suspension. The next day, 10 µl of 10 x concentrated test compound solutions were added. The effect of helquats was investigated in concentration range 1 to 100 µmol.l-1 (e.g. 1; 2.5; 5; 7.5; 10; 15; 25; 50 and 100 µmol.l-1). Apart from the section containing the concentration series of the test compound, each well contained 2 control columns, the first with pure medium (so called blank) and the second with cells in medium without the test compounds (control). Into wells containing controls and blanks, volume of solvent (water) equal to the volume of added compound solutions was added. After 72 h of incubation with the test compounds viability was determined according to the manufacturer's instructions. In short: microtiter well plate was left 30 min to reach the room temperature after it has been removed from the incubator. Then, into each well, 100 µl of the prepared detection agent was added. Next, the well plate was shaken for 2 min to complete the full lysis. Next, after the well had been left in the dark for 15 min, the luminescence was measured in each well using luminometer (Tecan Genios, Austria).
Table 1 Effect of helquats according to the invention, on cancer cells CCRF-CEM, MOLT-4, HeLa, HL-60, LoVo, Hep G2 and normal (non-cancer) cells HUVEC and NHDF-Ad CCRF-CEM MOLT-4 HeLa HepG2 HL-60 LoVo HUVEC NHDF-Ad (rac)-1 ** ** ** - - - >150 * (M)-1 *** *** *** ** *** ** >150 * (P)-1 ** ** ** - - - >150 * (rac)-7 *** ** ** - - - >150 >150 (M)-7 *** *** *** ** *** ** * * (P)-7 ** ** ** - - - * * (rac)-25 ** ** ** - - * >150 >150 30 * * * - - >150 >150 >150 (rac)-35 * * * - - >150 >150 >150 (rac)-41 ** ** ** - - * * * (rac)-20 ** ** ** - - * * * (M)-6 ** ** ** ** ** ** >150 >150 (rac)-12 ** * ** ** - * * * *** IU50 = 0-10 µmol.l-1
** IC50 = 11-50 µmol.l-1
* IC50 = 51-150 µmol.l-1
- IC50 value was not determined - IC50 value represents concentration of the test compound, which leads to 50% reduction of viable cells (inhibition of cell growth) after 72 h of the treatment. Each helquat concentration was tested in triplicates in a IC50 quantification and at least 3 independent IC50 quantification experiments were performed. IC50 values over 100 µmol.l-1 were obtained by extrapolation of data measured in concentration range 0 - 100 µmol.l-1 for the given helquat.
- The invention is useful in pharmaceutical industry and medicine for the treatment of diseases related to increased cellular proliferation, for example tumor growth.
Claims (11)
- Helquat derivatives of general formula I
substituents R1 and R2 are independently selected from the group comprising H and C1 to C3 alkyl,up to three of S1,2, S1',2', S3,4 and S3',4' are present,S1,2, S1',2', S3,4 and S3',4' independently represent a linker consisting of a bivalent hydrocarbon chain having 3-6 carbon atoms, andone or two atoms selected from the carbon atoms with the descriptor 2, 4, 2', and 4'are substituted with a substituent R3 of general formula II
T1 and T2 independently represent a bivalent hydrocarbon chain having 2-5 carbon atoms,
wherein
aryl is a hydrocarbon group containing 6 to 16 carbon atoms and, and at least one aromatic ring, wherein the aryl can be unsubstituted or substituted with 1 to 5 substituents, selected from a group comprising C1 to C6 alkyl, C1 to C6 halogenoalkyl, C1 to C12 alkoxy, aryloxy, benzyloxy, C1 to C6 alkylthio, arylthio, halogeno, -OH, -SH,-NH2, C1 to C6 alkylamino, arylamino, C1 to C6 acylamino, -CN, nitro, and -COORn, wherein Rn is hydrogen or C1 to C6 alkyl or aryl;and anions (X1)- and (X2)- independently represent anions of pharmaceutically acceptable salts of general formula I. - Helquat derivatives according to claim 1 selected from:(rac)-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(M-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(P)-(E)-13-(4-(dimethylamino)styryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(rac)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino-[1,2-a]pyrido[1,2-k] [2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(M)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino- [1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(P)-(E)-13-(2-(6-methoxynaphthalene-2-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino- [1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(rac)-(E)-11-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino[1,2-a]pyrido[1,2-k][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(rac)-(E)-13-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-4,5,8,9-tetrahydro-isoquinolino[2,1-k]pyrido[2,1-a][2,9]phenanthroline-3,10-diium trifluoromethanesulfonate,(rac)-(E)-19-(2-(1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)vinyl)-8,9,10,13,14,15-hexahydropyrido[1"',2'':1",2"]azepino[4",3":5',6']benzo[1',2':3,4]-azepino[2,1-a]isoquinoline-7,16-diium trifluoromethanesulfonate, 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-6,7,10,11-tetrahydrodipyrido [2,1-a:1',2'-k] [2,9]phenanthroline-5,12-diium trifluoromethanesulfonate,(M)-(E)-13-(4-methoxystyryl)-6,7-dimethyl-4,5,8,9-tetrahydroisoquinolino[1,2-a]pyrido-[1,2-k][2,9]phenanthroline-3,10-diiumtrifluoromethansulfonate, and(rac)-4,15-bis((E)-4-(dimethylamino)styryl)-6,7,8,11,12,13 -hexahydrodipyrido-[1,2-a':1',2'-a']benzo[2,1-c:3,4-c']bisazepinediium trifluoromethanesulfonate.
- Helquat derivatives of general formula I according to claim 1 for use as medicaments.
- Helquat derivatives of general formula I according to claim 1 for use as medicaments in the treatment of diseases related to increased cellular proliferation.
- Helquat derivatives of general formula I according to claim 1 for use in the treatment of oncologic diseases.
- A method of preparation of helquat derivatives of general formula I as described in claim 1, wherein a starting helquat bearing reactive methyl group is reacted with substituted or unsubstituted arylaldehyde in the presence of a base, preferably pyrrolidine or piperidine, and in an organic solvent, and the resulting product is isolated.
- The method of preparation according to claim 6, wherein a solvent, selected from methanol, ethanol, acetonitrile, dimethylsulfoxide and dimethylformamide, is used as the organic solvent.
- A pharmaceutical agent, containing at least one helquat derivative of general formula I according to claim 1 or its pharmaceutically acceptable salt.
- The pharmaceutical agent according to claim 8, which further contains at least one pharmaceutically acceptable carrier, filler, or diluent.
- The pharmaceutical agent according to claim 8 or 9 for use in the treatment of diseases related to increased cellular proliferation.
- The pharmaceutical agent according to claim 8 or 9 for use in the treatment of oncologic diseases.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CZ2013-32A CZ304996B6 (en) | 2013-01-17 | 2013-01-17 | Helquat derivatives, their preparation process and their use as medicaments |
| PCT/CZ2014/000009 WO2014111069A1 (en) | 2013-01-17 | 2014-01-17 | Helquat derivatives, preparation thereof, and use thereof as medicaments |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP2945937A1 EP2945937A1 (en) | 2015-11-25 |
| EP2945937B1 true EP2945937B1 (en) | 2017-02-22 |
Family
ID=50064318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP14702744.5A Not-in-force EP2945937B1 (en) | 2013-01-17 | 2014-01-17 | Helquat derivatives, preparation thereof, and use thereof as medicaments |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US9340543B2 (en) |
| EP (1) | EP2945937B1 (en) |
| AU (1) | AU2014207125B2 (en) |
| CA (1) | CA2895539C (en) |
| CZ (1) | CZ304996B6 (en) |
| WO (1) | WO2014111069A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ307163B6 (en) * | 2014-05-29 | 2018-02-14 | Ústav organické chemie a biochemie AV ČR, v.v.i. | Helquats with heteroaromatic substituents, preparation thereof, and use thereof as G-quadruplex stabilizers |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPS234402A0 (en) * | 2002-05-15 | 2002-06-13 | Auckland Uniservices Limited | Anti-tumour polycyclic carboxamides |
| US20090215908A1 (en) * | 2007-09-24 | 2009-08-27 | Reliance Life Sciences Pvt. Ltd. | Toll like receptor (tlr) signaling antagonist |
| CZ2009237A3 (en) * | 2009-04-16 | 2010-10-27 | Ústav organické chemie a biochemie Akademie ved Ceské republiky, v. v. i. | Novel helquats, precursors thereof and process of their preparation |
-
2013
- 2013-01-17 CZ CZ2013-32A patent/CZ304996B6/en not_active IP Right Cessation
-
2014
- 2014-01-17 US US14/655,999 patent/US9340543B2/en active Active
- 2014-01-17 CA CA2895539A patent/CA2895539C/en not_active Expired - Fee Related
- 2014-01-17 EP EP14702744.5A patent/EP2945937B1/en not_active Not-in-force
- 2014-01-17 WO PCT/CZ2014/000009 patent/WO2014111069A1/en active Application Filing
- 2014-01-17 AU AU2014207125A patent/AU2014207125B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| AU2014207125B2 (en) | 2016-06-02 |
| WO2014111069A1 (en) | 2014-07-24 |
| US9340543B2 (en) | 2016-05-17 |
| CZ304996B6 (en) | 2015-03-18 |
| AU2014207125A1 (en) | 2015-07-09 |
| CA2895539A1 (en) | 2014-07-24 |
| US20150344477A1 (en) | 2015-12-03 |
| CA2895539C (en) | 2016-09-06 |
| CZ201332A3 (en) | 2014-07-30 |
| EP2945937A1 (en) | 2015-11-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP4112606A1 (en) | Aromatic compound and use thereof in preparing antineoplastic drugs | |
| CA2883068C (en) | Metal-based thiophene photodynamic compounds and their use | |
| EP3712129A1 (en) | Alpha, beta-unsaturated amide compound | |
| EP3782995B1 (en) | Spiro condensed imidazoline-2,4-dione derivatives as histone acetyltransferase inhibitors for the treatment of cancer diseases | |
| JP4805166B2 (en) | Aroylfuran and aroylthiophene | |
| Valderrama et al. | Studies on quinones. Part 41: Synthesis and cytotoxicity of isoquinoline-containing polycyclic quinones | |
| Martin-Encinas et al. | Fused chromeno and quinolino [1, 8] naphthyridines: Synthesis and biological evaluation as topoisomerase I inhibitors and antiproliferative agents | |
| Abouelenein et al. | Synthesis, DFT calculations, In silico studies, and biological evaluation of pyrano [2, 3-c] pyrazole and pyrazolo [4′, 3′: 5, 6] pyrano [2, 3‐d] pyrimidine derivatives | |
| Loza-Mejía et al. | Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure–activity relationship of 9-anilinothiazolo [5, 4-b] quinoline derivatives | |
| EP2945937B1 (en) | Helquat derivatives, preparation thereof, and use thereof as medicaments | |
| Haroutounian et al. | Hydroxystilbazoles and hydroxyazaphenanthrenes: photocyclization and fluorescence studies | |
| CN107573318A (en) | A kind of new gossypol Schiff bases derivative and its synthetic method for having antitumor activity | |
| US11786520B2 (en) | Method for producing benzazoloquinolium (BQs) salts and using the biological activity of the composition | |
| CN104059062A (en) | Benzothiazole and triazolediheterocycle-containing fused ring compound and application thereof | |
| EP4215521A1 (en) | Fused ring diimide derivative, preparation method therefor and use thereof | |
| KR102606167B1 (en) | Fluorine-containing substituted benzothiophene compounds, pharmaceutical compositions and applications thereof | |
| CN116096372A (en) | EGFR inhibitor, preparation method and pharmaceutical application thereof | |
| CN114276330B (en) | Piperidone compound and preparation method and application thereof | |
| WO2004083228A1 (en) | Indolopyrrolocarbazole derivatives and antitumor agents | |
| Zhang et al. | Synthesis, in Vitro and in Vivo Anticancer Activity of Hybrids of 3-Hydroxy-indolin-2-one and 2, 3-Dihydroquinolin-4 (1H)-one | |
| CN110183471B (en) | Piperazine derivative, preparation method and application | |
| EP1758904B1 (en) | Flavopereirine derivatives for cancer therapy | |
| EP4085059B1 (en) | Heteroaryl compounds as inhibitors of programmed necrosis pathway, composition and method using the same | |
| Tylińska et al. | Synthesis and Anticancer Activity of New 1‐Substituted‐6H‐pyrido [4, 3‐b] carbazole Derivatives | |
| RU2704326C1 (en) | N-methoxybenzyl derivatives of daunorubicin having antiproliferative properties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 20150623 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
| AX | Request for extension of the european patent |
Extension state: BA ME |
|
| DAX | Request for extension of the european patent (deleted) | ||
| REG | Reference to a national code |
Ref country code: DE Ref legal event code: R079 Ref document number: 602014006908 Country of ref document: DE Free format text: PREVIOUS MAIN CLASS: C07D0401100000 Ipc: C07D0471220000 |
|
| RIC1 | Information provided on ipc code assigned before grant |
Ipc: C07D 471/22 20060101AFI20160713BHEP Ipc: A61P 35/00 20060101ALI20160713BHEP Ipc: A61K 31/435 20060101ALI20160713BHEP |
|
| GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
| INTG | Intention to grant announced |
Effective date: 20160826 |
|
| GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
| GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
| AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
| REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
| REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
| REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 869161 Country of ref document: AT Kind code of ref document: T Effective date: 20170315 |
|
| REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
| REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602014006908 Country of ref document: DE |
|
| REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG4D |
|
| REG | Reference to a national code |
Ref country code: NL Ref legal event code: MP Effective date: 20170222 |
|
| REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 869161 Country of ref document: AT Kind code of ref document: T Effective date: 20170222 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170522 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170523 Ref country code: HR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: RS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: NL Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20170222 Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170622 Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170522 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602014006908 Country of ref document: DE |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: PL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
| REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 5 |
|
| 26N | No opposition filed |
Effective date: 20171123 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| REG | Reference to a national code |
Ref country code: BE Ref legal event code: MM Effective date: 20180131 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20180131 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MT Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20180117 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20200120 Year of fee payment: 7 Ref country code: DE Payment date: 20200121 Year of fee payment: 7 Ref country code: IE Payment date: 20200116 Year of fee payment: 7 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: LU Payment date: 20200120 Year of fee payment: 7 Ref country code: CH Payment date: 20200120 Year of fee payment: 7 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MK Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20170222 Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20140117 Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20200116 Year of fee payment: 7 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170622 Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170222 |
|
| REG | Reference to a national code |
Ref country code: DE Ref legal event code: R119 Ref document number: 602014006908 Country of ref document: DE |
|
| REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
| GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20210117 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210117 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210131 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210131 Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210131 Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210117 Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210803 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20210117 |